Count following trim.seqs is way down


I’m familiar with mothur. I have used it on a number of sff files. I’ve encountered a problem with a particular sff and oligo file. Following trim.seqs the total count is only 395 reads. When I modify the oligo file and submit it along with the sff file to the RDP website for “Pipeline Initial Process” I get back over 450,000 reads.

During the shhh.flows command I noticed that the majority of the files have an error as seen here:

Processing plate.G6208A.flow (file 1 of 82) <<<<<
Reading flowgrams…
Identifying unique flowgrams…
Calculating distances between flowgrams…

Total time: 0 0

Clustering flowgrams…
[ERROR]: plate.G6208A.shhh.dist is blank. Please correct.
Reading matrix: |||||||||||||||||||||||||||||||||||||||||||||||||||

Does anyone have an idea what is going on? I appreciate the help.


This generally happens because the barcode was not found in the trim.flows step. You might double check that you have your barcode sequences correct

Hi Pat and bfh6152,
I believe to have the same problem, or at least I have the same error message:
mothur > shhh.flows(flow=/Applications/mothurGUI/HD34BBB.MID20.flow)

Using 1 processors.

Processing /Applications/mothurGUI/HD34BBB.MID20.flow (file 1 of 1) <<<<<
Reading flowgrams…
Identifying unique flowgrams…
Calculating distances between flowgrams…
0 0 7e-06
56 0 0.002248

Total time: 0 0.038383

Clustering flowgrams…
[ERROR]: /Applications/mothurGUI/HD34BBB.MID20.shhh.dist is blank. Please correct.

I actually do not even have the "reading matrix" part. Pat, I checked and rechecked the barcodes, there are good, I also tried with different oligo files. The problem came while running sff.multiple. It ran very well for most of the samples, and then for this one it does not want to complete the shhh.flow part. I tried also using the "non-multiple" pipeline, focusing only on this sample, it does not work... I checked the flow file, there is something inside.

Any idea?


So this sample only has 56 sequences in it and it looks like they must all be different from each other meaning that they can’t be denoised. Can you do anything with 56 sequences in a sample?

ok, I feel stupid now…
I am really beginner in that and I did not even checked…
Thanks for your help.